The present invention relates to an antibody fragment capable of binding to P-glycoprotein associated with multidrug resistant (MDR) cells. The present invention also relates to compositions and methods utilizing such an antibody fragment for inhibiting drug efflux activity in MDR cancer cells.
Cancer chemotherapy often fails due to the development of acquired or intrinsic resistance in cancerous cells to a wide variety of anticancer drugs, such as colchicine, vinblastine, vincristine and doxorubicin. This phenomenon, which is known as multidrug resistance (MDR), is a major barrier to cancer chemotherapy.
A key mechanism of MDR is the overexpression of an energy-dependant efflux pump, known as the multidrug transporter. This efflux pump is a 170 kDa P-glycoprotein (Pgp), encoded by the MDR1 gene. Pgp-mediated MDR plays an important role in the resistance of various tumor cells to chemotherapy; studies have shown a clear correlation between mdr1 expression and the lack of response to chemotherapy (6,7).
Inhibitors of the MDR phenotype in cancer cells may either modify or disrupt the expression of the drug efflux function of the transporter proteins involved in MDR. Known MDR inhibitors include verpamil (a calcium-channel blocker and being used to treat leukemia), cyclosporins, steroids, and calmodulin inhibitors (which enhance the intracellular accumulation and cytotoxic action of Pgp-transported drugs). However, most known MDR-modulating drugs presently available for clinical application have major side effects which substantially limit their therapeutic value.
Recently, Pgp-specific monoclonal antibodies (Mab) have been developed as intended agents for use in MDR inhibition. U.S. Pat. No. 4,837,306 describes antibodies directed against the C-terminal portion of the intracellular domain of Pgp. These antibodies are not known to have an inhibitory effect on the drug efflux activity in MDR cells.
U.S. Pat. No. 5,766,946 describes a monoclonal antibody termed MM.17 that recognizes an epitope located on the forth extracellular loop of human Pgp. The MM.17 antibody was generated by immunizing mice with an MDR variant of a human T-lymphoblastoid. This antibody is not known to have an inhibitory effect on the drug efflux activity in MDR cells.
U.S. Pat. No. 6,479,639 describes a monoclonal antibody termed UIC2 directed against an extracellular domain of Pgp. The UIC2 antibody was generated by immunizing mice with transfected fibroblast cells expressing Pgp. The UIC2 Mab was found capable of inhibiting the drug efflux activity of MDR cells in vitro.
Monoclonal antibodies termed HYB-241 and HYB-612, recognize an external epitope of Pgp. These Mabs have been reported to increase the accumulation of the chemotherapeutic drugs vincristine and actinomycin D in tumor cells thereby increasing cytotoxicity [Meyers, M. B. et al., Cancer Res., 49:3209 (1987); O'Brien, J. P. et al., Proc. Amer. Assoc. Cancer Res., 30:Abs 2114 (1989)].
The monoclonal antibody Mab657 has been reported to react with MDR cells [Cinciarelli, C., et al., Int. J. Cancer, 47:533 (1991)]. This antibody was reported to increase the susceptibility of MDR cells to cytotoxicity mediated by human peripheral blood lymphocytes, it is not known to have an inhibitory effect on the drug efflux activity of Pgp.
The monoclonal antibodies MRK-16 and MRK-17, were generated by immunizing mice with MDR human leukemia cells. Both antibodies recognize Pgp and are capable of modulated the drug efflux activity in MDR cells MDR in vitro and in vivo [Hamada H., et al., Cancer Res. PNAS 83:7785 (1986); Pearson, J. W., et al., J. Natl. Cancer Inst. 88:1386 (1991); Tsuruo, T., et. al., Jpn. J. Cancer Res. 80:627 (1989)]. A recombinant chimeric antibody that combines the variable region of MRK-16 with the Fc portion of human antibodies was reported to be more effective than parent MRK-16 Mab in increasing cytotoxicity to MDR cells in vitro [Hamada H. et al., cancer Res. 50:3167 (1990)].
A substantial limitation of the above described antibodies stems from the large size of these molecules. It is well known that delivery efficiency of an agent is typically inversely proportional to its size. Thus, the large antibody molecules described above would not efficiently penetrate and distribute within the tumor tissue requiring high administration dosages to obtain therapeutic effect.
Another major shortcoming of presently available Pgp-specific antibodies is the fact that Pgp is constitutively expressed in normal human tissues, including kidney, liver, colon, testis, lymphocytes, and the blood-brain barrier (27). A large antibody molecule would typically circulate for extended time periods and would be slowly cleared from circulation resulting in possible toxic effects on normal tissues that physiologically express Pgp.
In addition, most of the Pgp-specific antibodies described above were selected for high affinity to Pgp. However, the practical use of Pgp-specific antibodies in therapy may be substantially restricted when the binding affinity of the antibody to Pgp is high. This is due to toxic effects that might be exerted on normal tissues that physiologically express Pgp.
There is thus a widely recognized need for, and it would be highly advantageous to have, a Pgp-specific antibody devoid of the above limitations.